Estimation of the bacteriocin ColE7 conjugation-based "kill" - "anti-kill" antimicrobial system by real-time PCR, fluorescence staining and bioluminescence assays.

The efficiency of the bacteriocin, colicin ColE7, bacterial conjugation-based "kill" - "anti-kill" antimicrobial system, was assessed using real-time PCR, flow cytometry and bioluminescence. The ColE7 antimicrobial system consists of the genetically modified Escherichia coli strain Nissle 1917 harbouring a conjugative plasmid (derivative of the F-plasmid) encoding the "kill" gene (ColE7 activity gene) and a chromosomally encoded "anti-kill" gene (ColE7 immunity gene). On the basis of traJ gene expression in the killer donor cells, our results showed that the efficiency of the here studied antimicrobial system against target E. coli was higher at 4 than at 24 h. Flow cytometry was used to indirectly estimate DNase activity of the antimicrobial system, as lysis of target E. coli cells in the conjugative mixture with the killer donor strain led to reduction in cell cytosol fluorescence. According to a lux assay, E. coli TG1 (pXen lux+ Apr ) with constitutive luminescence were killed already after 2 h of treatment. Target sensor E. coli C600 with DNA damage SOS-inducible luminescence showed significantly lower SOS induction 6 and 24 h following treatment with the killer donor strain. Our results thus showed that bioluminescent techniques are quick and suitable for estimation of the ColE7 bacterial conjugation-based antimicrobial system antibacterial activity. SIGNIFICANCE AND IMPACT OF THE STUDY: Bacterial antimicrobial resistance is worldwide rising and causing deaths of thousands of patients infected with multi-drug resistant bacterial strains. In addition, there is a lack of efficient alternative antimicrobial agents. The significance of our research is the use of a number of methods (real-time PCR, flow cytometry and bioluminescence-based technique) to assess the antibacterial activity of the bacteriocin, colicin ColE7, bacterial conjugation-based "kill" - "anti-kill" antimicrobial system. Bioluminescent techniques proved to be rapid and suitable for estimation of antibacterial activity of ColE7 bacterial conjugation-based antimicrobial system and possibly other related systems.

In vivo EPR on spin labeled colicin A reveals an oligomeric assembly of the pore-forming domain in E. coli membranes.

We report on the application of site-directed spin labeling (SDSL) and electron paramagnetic resonance (EPR) spectroscopy to study possible oligomerization of the bacterial toxin colicin A (ColA) upon membrane insertion in vitro and in vivo. We applied SDSL-EPR protocols and optimized experimental conditions to perform continuous wave EPR experiments and double electron-electron resonance distance measurements on intact Escherichia coli cells interacting with nitroxide spin-labeled ColA. Our data suggest that ColA forms dimers upon membrane insertion, thus explaining previously reported pore diameters of about 1 nm, which are unlikely to be formed by a single colicin A monomer.

[Detection of uncultivable bacteria forms in probiotic lyophilized preparations].

AIM: Detection of viable but uncultivable forms among lyophilized cells of commercial probiotics. MATERIALS AND METHODS: 9 series of probiotic preparations (colibacterin, bificol, bifidumbacterin, bifiform) with expired (up to 30 years of storage) or valid shelf life were objects of the study. Total quantity of the bacteria was calculated under the microscope in Goryaev chamber, the number of viable cells was determined in luminescence microscope after staining by an array of fluorescent dyes, the quantity of CFU/ml was evaluated by method of seeding into the respective solid or semi-liquid cultivation medium. RESULTS: Juxtaposition of the specified parameter values allowed to establish that in lyophilized preparations of colibacterin with expired shelf life the amount of viable but not forming colonies (uncultivable forms) cells varied from 4.13 to 99.73% depending on date of production. For bifidumbacterin with unexpired shelf life live cells constituted 95.45 and 70.73%. The amount of viable bifidobacteria forming colonies was on the level of 100 and 50%, respectively. In bifidopreparations micro colonies that may possibly be formed spontaneously by awakened uncultivable forms were noted. A possibility of growth stimulation of these cells by 1 and 10% aminopeptide was shown. CONCLUSION: The presence of uncultivable forms in lyophilized preparations of probiotics was proven experimentally.

Molecular and antimicrobial susceptibility analyses distinguish clinical from bovine Escherichia coli O157 strains.

A population-based study combining (i) antimicrobial, (ii) genetic, and (iii) virulence analyses with molecular evolutionary analyses revealed segregative characteristics distinguishing human clinical and bovine Escherichia coli O157 strains from western Canada. Human (n = 50) and bovine (n = 50) strains of E. coli O157 were collected from Saskatchewan and Manitoba in 2006 and were analyzed by using the six-marker lineage-specific polymorphism assay (LSPA6), antimicrobial susceptibility analysis, the colicin assay, plasmid and virulence profiling including the eae, ehxA, espA, iha, stx1, stx2, stx2c, stx2d, stx2d-activatable, stx2e, and stx2f virulence-associated genes, and structure analyses. Multivariate logistic regression and Fisher's exact test strongly suggested that antimicrobial susceptibility was the most distinctive characteristic (P = 0.00487) associated with human strains. Among all genetic, virulence, and antimicrobial determinants, resistance to tetracycline (P < 0.000) and to sulfisoxazole (P < 0.009) were the most strongly associated segregative characteristics of bovine E. coli O157 strains. Among 11 virulence-associated genes, stx2c showed the strongest association with E. coli O157 strains of bovine origin. LSPA6 genotyping showed the dominance of the lineage I genotype among clinical (90%) and bovine (70%) strains, indicating the importance of lineage I in O157 epidemiology and ecology. Population structure analysis revealed that the more-diverse bovine strains came from a unique group of strains characterized by a high degree of antimicrobial resistance and high frequencies of lineage II genotypes and stx2c variants. These findings imply that antimicrobial resistance generated among bovine strains of E. coli O157 has a large impact on the population of this human pathogen.

Burkholderia cenocepacia requires a periplasmic HtrA protease for growth under thermal and osmotic stress and for survival in vivo.

Burkholderia cenocepacia, a member of the B. cepacia complex, is an opportunistic pathogen that causes serious infections in patients with cystic fibrosis. We identified a six-gene cluster in chromosome 1 encoding a two-component regulatory system (BCAL2831 and BCAL2830) and an HtrA protease (BCAL2829) hypothesized to play a role in the B. cenocepacia stress response. Reverse transcriptase PCR analysis of these six genes confirmed they are cotranscribed and comprise an operon. Genes in this operon, including htrA, were insertionally inactivated by recombination with a newly created suicide plasmid, pGPOmegaTp. Genetic analyses and complementation studies revealed that HtrA(BCAL2829) was required for growth of B. cenocepacia upon exposure to osmotic stress (NaCl or KCl) and thermal stress (44 degrees C). In addition, replacement of the serine residue in the active site with alanine (S245A) and deletion of the HtrA(BCAL2829) PDZ domains demonstrated that these areas are required for protein function. HtrA(BCAL2829) also localizes to the periplasmic compartment, as shown by Western blot analysis and a colicin V reporter assay. Using the rat agar bead model of chronic lung infection, we also demonstrated that inactivation of the htrA gene is associated with a bacterial survival defect in vivo. Together, our data demonstrate that HtrA(BCAL2829) is a virulence factor in B. cenocepacia.

[Amplified fragment length polymorphism genotyping of Shigellae and comparison to pulsed-field gel electrophoresis and colicin typing].

Shigella is an etiological agent of communicable and food-borne disease worldwide, so it is important to develop typing for Shigella in epidemiological studies. We compared amplified fragment length polymorphism (AFLP), molecular epidemiological typing, to pulsed-field gel electrophoresis (PFGE) and colicin typing in typeability, reproducibility, discriminatory power, ease of interpretation, and ease of use for 51 Shigella isolates to determine AFLP applicability to Shigella. AFLP showed less reproducibility and ease of interpretation although it was superior to PFGE and colicin typing in typeability and discriminatory power. Specifying the reproducibility of these typing methods, the intrastrain similarity of AFLP was 81.9%-90.5% in each of three strains tested in triplicate trials, while PFGE showed higher similarity, ranging from 92.3%-100%. AFLP created a phylogenetic tree and classified four Shigella species taxonomically, despite its lower reproducibility. These results suggest that AFLP is inferior to PFGE as molecular typing for Shigella epidemiologically in outbreaks or sporadic cases, although AFLP can create a phylogenetic tree for taxonomical purposes.

Proteomic analysis of extracellular proteins from Escherichia coli W3110.

While numerous proteomic analyses have been carried out on Escherichia coli, the vast majority have focused on expression of intracellular proteins. Yet, recent literature reports imply that even in laboratory strains, significant proteins may be found outside the cell. Here, we identify extracellular proteins associated with nonpathogenic E. coli strain W3110. Two-dimensional gel electrophoresis (2DE) revealed approximately 66 prominent protein spots during exponential growth (4 and 8 h shake flask culture) in minimal medium. The absence of detectable nucleic acids in the culture supernatant implies these proteins did not result from cell lysis. MALDI-TOF MS was used to identify 44 proteins, most of which have been previously identified as either outer membrane or extracellular proteins. In addition, 2DE protease zymogram analysis was carried out which facilitated identification of three extracellular proteases, one of which was not observed during standard 2DE. Our results are consistent with previous findings which imply outer membrane proteins are shed during growth.

Exoproducts of the Escherichia coli strain H22 inhibiting some enteric pathogens both in vitro and in vivo.

AIMS: The antagonistic activity of the Escherichia coli strain H22 against enteric bacteria was studied both in vitro and in vivo. METHODS AND RESULTS: In vitro, bacterial strains belonging to seven of nine genera of the family Enterobacteriaceae (Enterobacter, Escherichia, Klebsiella, Morganella, Salmonella, Shigella and Yersinia) were inhibited by the strain H22. Six days after simultaneous oral inoculation in germ-free mice, E. coli strain H22 reduced the faecal population of Shigella flexneri 4 to undetectable levels (P < 0.05). In ex vivo assay, inhibitory zones against Sh. flexneri 4 were observed around faecal samples from mice inoculated with E. coli strain H22. The in vitro inhibition of Sh. flexneri 4 was shown to be mediated by microcin C7. In addition to microcin C7, strain H22 was shown to produce aerobactin, new variants of colicins E1 and Ib, and bacteriophage particles with morphology similar to the phages of the family Myoviridae. CONCLUSIONS: Altogether, the properties of E. coli H22, observed both under in vitro and in vivo conditions, suggest its potential use as a probiotic strain for livestock and humans. SIGNIFICANCE AND IMPACT OF THE STUDY: The strain H22 was shown to produce several antimicrobial compounds with inhibitory capabilities against pathogenic or potentially pathogenic enterobacteria.

A 1D sensitivity-enhanced 1H spin diffusion experiment for determining membrane protein topology.

A sensitivity-enhanced 1D (1)H spin diffusion experiment, CHH, for determining membrane protein topology is introduced. By transferring the magnetization of the labeled protein (13)C to lipid and water protons for detection, the CHH experiment reduces the time of the original 2D (13)C-detected experiment by two orders of magnitude. The sensitivity enhancement results from (1)H detection and the elimination of the (13)C dimension. Consideration of the spin statistics of the membrane sample indicates that the CHH sensitivity depends on the (13)C labeling level and the number of protein protons relative to the mobile protons. 5-35% of the theoretical sensitivity was achieved on two extensively (13)C labeled proteins. The experimental uncertainties arise from incomplete suppression of the equilibrium (1)H magnetization and the magnetization of lipid protons directly bonded to natural-abundance carbons. The technique, demonstrated on colicin Ia channel domain, confirms the presence of a transmembrane domain and the predominance of surface-bound helices.

Colicin S8 export: extracellular and cytoplasmic colicin are different

The properties of colicin S8 are different for the cytoplasmic, periplasmic and extracellular protein. Interactions with its specific receptors reflect this. Active cell extracts separate into a non-anionic along with an anionic fraction by DEAE-Sephacell chromatography. Previously, we have purified cell-associated colicin S8 as an aggregation of highly related polypeptides; cytoplasmic colicin S8 seems to be post-translationally processed into an aggregation of polypeptides of molecular mass ranging from 45,000 Da to 60,000 Da. We suggest that a conformational change to colicin S8 may occur related to the export process (AU)

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Three recently acknowledged Escherichia species strikingly differ in the incidence of bacteriocinogenic and lysogenic strains.

The incidence of bacteriocinogeny and lysogeny was followed in bacteria of 3 recently acknowledged species of the genus Escherichia: E. hermanii, E. vulneris and E. fergusonii. Almost all of the strains examined were of human origin. In 30 strains of E. hermanii no one was found bacteriocinogenic while 57% were lysogenic, in 30 strains of E. vulneris none was found to be bacteriocinogenic and only 10% were lysogenic, and in 50 strains E. fergusonii 12% were bacteriocinogenic and 40% lysogenic. From the 6 bacteriocinogenic strains of E. fergusonii, 3 were producers of colicin E1, 2 of colicin Ib and 1 of colicin Ia. In addition, 3 E. fergusonii strains produced aerobactin.

[Colicinogenic activity of intestinal microflora as a sign of dysbacteriosis of the gastrointestinal tract]. / Kolitsinogennaia aktivnost' kishechnoi mikroflory kak pokazatel' disbioticheskogo sostoianiia zheludochno-kishechnogo trakta.

Clinical and bacteriological studies have revealed that the production of colicin by Escherichia coli forming a part of intestinal microbiocenosis is related to the clinical manifestations of inflammatory diseases of the gastrointestinal tract. During the exacerbation of chronic inflammatory processes of the digestive system the proportion of colicin producing E. coli increases (more than 45%) in comparison with that of E. coli fecal strains isolated in children and adolescents regarded as healthy (less than 15%). The possibility of using the colicin producing activity of intestinal microflora for the evaluation of the dysbiotic states of gastrointestinal tract is discussed.

Colicins produced by the Escherichia fergusonii strains closely resemble colicins encoded by Escherichia coli.

Plasmid DNA of six Escherichia fergusonii colicinogenic strains (three producers of colicin E1, two of Ib and one of Ia) was isolated and the colicin-encoding regions of the corresponding Col plasmids were sequenced. Two new variants of colicin E1, one of colicin Ib, and one of colicin Ia were identified as well as new variants of the colicin E1 and colicin Ib immunity proteins and the colicin E1 lysis polypeptide. The recombinant Escherichia coli producer harboring pColE1 from E. fergusonii strain EF36 (pColE1-EF36) was found to be only partially immune to E1 colicins produced by two other E. fergusonii strains suggesting that pColE1-EF36 may represent an ancestor ColE1 plasmid.

[Avian Escherichia coli virulence factors associated with coli septicemia in broiler chickens]. / Factores de virulencia de Escherichia coli aviar asociados a la colisepticemia en pollos de engorde.

In order to detect phenotypic characteristics associated with pathogenicity, 25 strains of Escherichia coli, isolated from clinical cases of colisepticemia in broiler chickens, were examined to determine the following properties: colicinogenicity, colicin V production, type 1 fimbriae, hemolysin expression and motility. Colicinogenicity occurred in 72% of the strains, 56% of all strains produced colicin V, 84% were positive for type 1 fimbriae and 80% were positive for motility. None of the strains had hemolytic activity; however, all of them, expressed at least one of the other characteristics studied. These results suggest that the diversity of phenotypes detected partially explain the multifactorial nature of avian colisepticemia.

Factores de virulencia de Escherichia coli aviar asociados a la colisepticemia en pollos de engorde / Avian Escherichia coli virulence factors associated with coli septicemia in broiler chickens

In order to detect phenotypic characteristics associated with pathogenicity, 25 strains of Escherichia coli, isolated from clinical cases of colisepticemia in broiler chickens, were examined to determine the following properties: colicinogenicity, colicin V production, type 1 fimbriae, hemolysin expression and motility. Colicinogenicity occurred in 72 of the strains, 56 of all strains produced colicin V, 84 were positive for type 1 fimbriae and 80 were positive for motility. None of the strains had hemolytic activity; however, all of them, expressed at least one of the other characteristics studied. These results suggest that the diversity of phenotypes detected partially explain the multifactorial nature of avian colisepticemia.(AU)

[Frequency of colicin and hemolysins in Escherichia coli isolated from pregnant patients with urinary tract infection, symptomatic and asymptomatic]. / Frecuencia de colicina y hemolisinas en Escherichia coli aisladas de pacientes embarazadas con infección de vías urinarias, sintomática y asintomática.

Colicin production was studied among 137 Escherichia coli strains isolated from pregnant patients with symptomatic or asymptomatic, urinary tract infection (IVU). The observed colicinogeny frequency was 72.92% (n = 96), among the symptomatic patients and 29.26% (n = 41) among the asymptomatic group. The most frequently identified colicins from symptomatic patients were: V (23.9%), A (18.7%) and E1 (17.7%) and within the patients with asymptomatic IVU the most frequently observed colicins were A (9.7%), E1 (17.0%) y V (2.4%). The major frequency of colicin V among the E. coli strains isolated from the symptomatic group against the asymptomatic one, was statistical significant (p > or = 0.025). The results on the observed frequencies of colicins E1 and A were not statistical significant. Among the 137 studied E. coli strains, 37.2% were hemolytic. 83.3% of the colicin V producing strains (n = 24) were hemolytic, among the strains producing other colicin different than colicin V 34% (n = 58) were hemolytic and 12.0% of non colicinogenic strains were hemolytic. These results were statistical significant (p > 0.05). The present data, suggest that colicins production is an important pathogenicity factor among the E. coli strains, specially for those strains producing colicin V. The observed association among hemolysin and colicin V production may be an interesting pathogenicity factor which suggests an increasing ability of uropathogenic strains to produce symptomatic urinary tract infections.

Estimation of the number of alpha-helical and beta-strand segments in proteins using circular dichroism spectroscopy.

A simple approach to estimate the number of alpha-helical and beta-strand segments from protein circular dichroism spectra is described. The alpha-helix and beta-sheet conformations in globular protein structures, assigned by DSSP and STRIDE algorithms, were divided into regular and distorted fractions by considering a certain number of terminal residues in a given alpha-helix or beta-strand segment to be distorted. The resulting secondary structure fractions for 29 reference proteins were used in the analyses of circular dichroism spectra by the SELCON method. From the performance indices of the analyses, we determined that, on an average, four residues per alpha-helix and two residues per beta-strand may be considered distorted in proteins. The number of alpha-helical and beta-strand segments and their average length in a given protein were estimated from the fraction of distorted alpha-helix and beta-strand conformations determined from the analysis of circular dichroism spectra. The statistical test for the reference protein set shows the high reliability of such a classification of protein secondary structure. The method was used to analyze the circular dichroism spectra of four additional proteins and the predicted structural characteristics agree with the crystal structure data.

Simplified methods for pKa and acid pH-dependent stability estimation in proteins: removing dielectric and counterion boundaries.

Much computational research aimed at understanding ionizable group interactions in proteins has focused on numerical solutions of the Poisson-Boltzmann (PB) equation, incorporating protein exclusion zones for solvent and counterions in a continuum model. Poor agreement with measured pKas and pH-dependent stabilities for a (protein, solvent) relative dielectric boundary of (4,80) has lead to the adoption of an intermediate (20,80) boundary. It is now shown that a simple Debye-Huckel (DH) calculation, removing both the low dielectric and counterion exclusion regions associated with protein, is equally effective in general pKa calculations. However, a broad-based discrepancy to measured pH-dependent stabilities is maintained in the absence of ionizable group interactions in the unfolded state. A simple model is introduced for these interactions, with a significantly improved match to experiment that suggests a potential utility in predicting and analyzing the acid pH-dependence of protein stability. The methods are applied to the relative pH-dependent stabilities of the pore-forming domains of colicins A and N. The results relate generally to the well-known preponderance of surface ionizable groups with solvent-mediated interactions. Although numerical PB solutions do not currently have a significant advantage for overall pKa estimations, development based on consideration of microscopic solvation energetics in tandem with the continuum model could combine the large deltapKas of a subset of ionizable groups with the overall robustness of the DH model.

Intracellular immunization of prokaryotic cells against a bacteriotoxin.

Intracellularly expressed antibodies have been designed to bind and inactivate target molecules inside eukaryotic cells. Here we report that an antibody fragment can be used to probe the periplasmic localization of the colicin A N-terminal domain. Colicins form voltage-gated ion channels in the inner membrane of Escherichia coli. To reach their target, they bind to a receptor located on the outer membrane and then are translocated through the envelope. The N-terminal domain of colicins is involved in the translocation step and therefore is thought to interact with proteins of the translocation system. To compete with this system, a single-chain variable fragment (scFv) directed against the N-terminal domain of the colicin A was synthesized and exported into the periplasmic space of E. coli. The periplasmic scFv inhibited the lethal activity of colicin A and had no effect on the lethal activity of other colicins. Moreover, the scFv was able to specifically inactivate hybrid colicins possessing the colicin A N-terminal domain without affecting their receptor binding. Hence, the periplasmic scFv prevents the translocation of colicin A and probably its interaction with import machinery. This indicates that the N-terminal domain of the toxin is accessible in the periplasm. Moreover, we show that production of antibody fragments to interfere with a biological function can be applied to prokaryotic systems.

Characterization of Salmonella dublin and Salmonella typhimurium (Copenhagen) isolates from cattle.

Eight Salmonella typhimurium (Copenhagen) and eight Salmonella dublin isolates from cattle were compared by their antibiotic resistance patterns, by their production of colicin, aerobactin, haemolysin and capsule, by their possession of transmissible R plasmids and the spvC gene, and by their ability to invade and replicate within cultured epithelial cells. The two groups differed in their antibiotic resistance profiles, with more of the host-adapted S. dublin isolates resistant to tetracycline than were the non-host-adapted S. typhimurium (Copenhagen) group, but more of the S. typhimurium (Copenhagen) isolates resistant to the other antibiotics tested. None of the isolates produced colicin, but all produced aerobactin. One isolate in each group was encapsulated. All of the S. typhimurium (Copenhagen) and S. dublin isolates contained plasmids, and all of them contained the spvC-homologous sequences. Four of the S. typhimurium (Copenhagen) isolates were able to transfer an R plasmid to a recipient organism by conjugation. One of the five S. dublin isolates, which showed resistance to some of the antibiotics tested, was able to transfer an R plasmid by conjugation. Both groups of isolates invaded cultured epithelial cells to a similar degree after 1 h, but the S. dublin isolates reached significantly higher levels within the cells than did S. typhimurium (Copenhagen) after 9 h. This ability may, in part, explain the association of S. dublin with more severe forms of salmonellosis and prolonged carrier states. Further study of the intracellular growth of these isolates seems warranted.